Proteasome inhibitors are now one of the first treatment options for multiple myeloma and are being evaluated in clinical trials against a variety of other cancers. However, these compounds, which target one of the three active sites of the proteasome, have drawbacks including side effects and emerging resistance. Therefore, it is important to develop and evaluate novel classes of proteasome inhibitors. Recently, we reported the discovery and characterization of the first pharmacological inhibitor of one of the ATPases of the 26S proteasome. These six different AAA class ATPases (called Rpts 1-6) are located at the base 19S regulatory particle (RP) that caps each end of the barrel-like 20S core particle (CP) of the proteasome. The ATPases constitute a protein unwinding activity that acts to introduce substrates into the catalytic interior of the 20S CP and also holds open a flap at the entrance that otherwise impedes substrate access. However, little is known about the contribution of each protein to the ATP-dependent unfolding process. Recent studies in yeast, many from our own laboratory, have also shown that these proteins play important, non-proteolytic roles in RNA polymerase II transcription, DNA repair and perhaps DNA replication. The individual contributions of each of the six ATPases to all of these activities are unclear. In this proposal, we intend to identify and optimize peptoid inhibitors of each of the six proteasomal ATPases. Large peptoid libraries will be synthesized and screened to identify hits using procedures and assays developed for the inhibitor mentioned above, which targets Rpt4. Rapid optimization procedures will be developed to improve the hits into nM leads. These compounds will then be utilized to determine if the proteasomal ATPases play a critical role in mammalian transcription, as is the case in yeast. The isolation of potent inhibitors of these proteins will then set the stage for future biochemical experiments to dissect the role of each protein in various steps in transcription and proteolysis. Future experiments would also include testing their activity in various mouse models of cancer.